Establishing a Co-Culture of Dental Pulp Cells and Trigeminal Neurons for Cross-Communication

After euthanization of the mouse, place the head on a disposable underpad so that the mouth is toward the ceiling and the base of the neck is flat on the work surface. Use a razor blade in a sawing motion to separate the mandible from the maxilla. Remove the tongue with scissors or forceps to allow easier access to the molars.

Place the opened head in a dish atop a sterile gauze pad, and place the specimen under a dissecting microscope. Remove the alveolar bone tissue surrounding the first molars. Insert forceps into the alveolar opening, and tease the tissue away from the tooth toward the buccal or lingual side of the mouth.

Collect all maxillary first molars and gently transfer the submandibular and maxillary first molars to a separate cell culture dish with 1X PBS on ice. Remove the enamel outer organs surrounding the outside of each maxillary first molar. With a set of forceps, rotate the molars so the cusps are down and the open root is exposed. There is an oval opening on the bottom of the tooth and opaque dental pulp tissue encapsulated by a thin layer of dentin and enamel.

Using the tip of the forceps, gently loosen the dental pulp by running one arm of the forceps around the internal circumference of the mineralized tissue. Remove the dental pulp tissue out of the mineralized structure, and transfer it to a third dish containing 1X PBS. Remove the enamel outer organ if it is not already separated.

Transfer all dental pulp tissue to 0.25% Trypsin EDTA in a 50-milliliter conical tube. Vortex the mixture and place it in a 37 degrees Celsius warm water bath for 10 minutes. Under a sterile hood, add warmed co-culture media to a final ratio of at least 1-to-1 media to trypsin to inactivate the enzyme. Pipette the media up and down multiple times with a 10-milliliter pipette to further disperse the dental pulp in the media. Avoid large bubbles.

Transfer 1 milliliter of the dispersed dental pulp to each well of a 24-well tissue culture plate. Place the plate in an incubator at 37 degrees Celsius, and allow the cells to attach and migrate out from the undisbursed tissue for 48 hours before changing media.

After euthanization and removal of skin, insert the tip of a pair of micro-dissecting scissors into the base of the skull. Cut along the sagittal suture of the skull. Make four small horizontal cuts– two along the coronal sutures by the ears and two along the lambdoid sutures at the base of the skull. This creates two flaps of bone.

Use the forceps to peel back the two flaps of bone to reveal the brain. Locate the trigeminal ganglia housed in the dura mater between the brain and bone of the maxillary process. Cut the three branches that travel to the eyes, maxillae, and mandible. and use straight-edge fine forceps to transfer the ganglia to cold 1X PBS in a dish on ice.

Once all trigeminal bundles are harvested, use vial forceps to transfer ganglia to a 50-milliliter conical tube containing 5 milligrams per milliliter of sterile, filtered collagenase type 2. Vortex, the mixture and place the tube in a 37 degrees Celsius water bath for 25 to 30 minutes. Every 5 to 10 minutes, take the tube out of the water bath, vortex, and return to the bath.

Centrifuge the collagenase trigeminal neuron solution for 2 minutes at 643 times g. Under a tissue culture hood, gently aspirate the collagenase with a micropipette and add 5 milliliters of 1% sterile filtered trypsin type 2.

Then, place the tube into a 37 degrees Celsius water bath for 25 to 30 minutes, and within this time frame, vortex to briefly every five minutes. Add media at a 1-to-1 ratio of trypsin to media to deactivate the remaining trypsin.

Count the number of cells and dilute the mixture to 200,000 cells per milliliter in media. Place coated transwell filters into wells with dental pulp. Pipette 250 microliters onto the transwell filter and cultured the cells at 37 degrees Celsius overnight.

The next day, replace the media with 1 milliliter of co-culture media supplemented with one micromolar uridine and 15 micromolar five fluoro-2-prime-deoxyuridine to stop the overproliferation of mesenchymal cells that may prevent neurite outgrowth.